Cells of the immune system are known to recognize and interact with specific molecules by means of receptors or receptor complexes which, upon recognition or an interaction with such molecules, causes activation of the cell to perform various functions. An example of such a receptor is the antigen-specific T cell receptor complex (TCR/CD3).
The T cell receptor for antigen (TCR) is responsible for the recognition of antigen associated with the major histocompatibility complex (MHC). The TCR expressed on the surface of T cells is associated with an invariant structure, CD3. CD3 is assumed to be responsible for intracellular signalling following occupancy of the TCR by ligand.
The T cell receptor for antigen-CD3 complex (TCR/CD3) recognizes antigenic peptides that are presented to it by the proteins of the major histocompatibility complex (MHC). Complexes of MHC and peptide are expressed on the surface of antigen presenting cells and other T cell targets. Stimulation of the TCR/CD3 complex results in activation of the T cell and a consequent antigen-specific immune response. The TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.
Two forms of T cell receptor for antigen are expressed on the surface of T cells. These contain either α/β heterodimers or γ/δ heterodimers. T cells are capable of rearranging the genes that encode the α, β, γ and δ chains of the T cell receptor. T cell receptor gene rearrangements are analogous to those that produce functional immunoglobulins in B cells and the presence of multiple variable and joining regions in the genome allows the generation of T cell receptors with a diverse range of binding specificities. Each α/β or γ/δ heterodimer is expressed on the surface of the T cell in association with four invariant peptides. These are the γ, Ε and ε subunits of the CD3 complex and the zeta chain. The CD3 γ, δ and ε polypeptides are encoded by three members of the immunoglobulin supergene family and are found in a cluster on human chromosome 11 or murine chromosome 9. The zeta chain gene is found separately from other TCR and CD3 genes on chromosome 1 in both the mouse and human. Murine T cells are able to generate a receptor-associated η chain through alternative splicing of the zeta m-RNA transcript. The CD3 chains and the zeta subunit do not show variability, and are not involved directly in antigen recognition.
All the components of the T cell receptor are membrane proteins and consist of a leader sequence, externally-disposed N-terminal extracellular domains, a single membrane-spanning domain, and cytoplasmic tails. The α, β, γ and δ antigen-binding polypeptides are glycoproteins. The zeta chain has a relatively short ectodomain of only nine amino acids and a long cytoplasmic tail of approximately 110 amino acids. Most T cell receptor α/β heterodimers are covalently linked through disulphide bonds, but many γ δ receptors associate with one another non-covalently. The zeta chain quantitatively forms either disulphide-linked ζ-η heterodimers or zeta-zeta homodimers.
Another example of a type of receptor on cells of the immune system is the Fc receptor. The interaction of antibody-antigen complexes with cells of the immune system results in a wide array of responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signals such as regulating lymphocyte proliferation, phagocytosis and target cell lysis. All these interactions are initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. It is now well established that the diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of Fc receptors (FcRs).
FcRs are defined by their specificity for immunoglobulin isotypes. Fc receptors for IgG are referred to as FcγR, for IgE as FcεR, for IgA as FcαR, etc. Structurally distinct receptors are distinguished by a Roman numeral, based on historical precedent. We now recognize three groups of FcγRs, designated FcγRI, FcγRII, and FcγRIII. Two groups of FcεR have been defined; these are referred to as FcεRI and FcεRII. Structurally related although distinct genes within a group are denoted by A, B, C. Finally, the protein subunit is given a Greek letter, such as FcγRIIIAα, FcγRIIIAγ.
Considerable progress has been made in the last three years in defining the heterogeneity for IgG and IgE Fc receptors (FcγR, FcεR) through their molecular cloning. Those studies make it apparent that Fc receptors share structurally related ligand binding domains, but differ in their transmembrane and intracellular domains which presumably mediate intracellular signalling. Thus, specific FcγRs on different cells mediate different cellular responses upon interaction with an immune complex. The structural analysis of the FcγRs and FcεRI has also revealed at least one common subunit among some of these receptors. This common subunit is the γ subunit, which is similar to the ζ or η chain of the TCR/CD3, and is involved in the signal transduction of the FcγRIII and FcεRI.
The low affinity receptor for IgG (FcγRIIIA), is composed of the ligand binding CD16α (FcγRIIIAα) polypeptide associated with the γ chain (FcγRIIIAγ). The CD16 polypeptide appears as membrane anchored form in polymorphonuclear cells and as transmembrane form (CD16TM) in NK. The FcγRIIIA serves as a triggering molecule for NK cells.
Another type of immune cell receptor is the IL-2 receptor. This receptor is composed of three chains, the α chain (p55), the β chain (p75) and the γ chain. When stimulated by IL-2, lymphocytes undergo proliferation and activation.
Antigen-specific effector lymphocytes, such as tumor specific T cells (Tc), are very rare, individual-specific, limited in their recognition spectrum and difficult to obtain against most malignancies. Antibodies, on the other hand, are readily obtainable, more easily derived, have wider spectrum and are not individual-specific. The major problem of applying specific antibodies for cancer immunotherapy lies in the inability of sufficient amounts of monoclonal antibodies (mAb) to reach large areas within solid tumors. In practice, many clinical attempts to recruit the humoral or cellular arms of the immune system for passive anti-tumor immunotherapy have not fulfilled expectations. While it has been possible to obtain anti-tumor antibodies, their therapeutic use has been limited so far to blood-borne tumors (1, 2) primarily because solid tumors are inaccessible to sufficient amounts of antibodies (3). The use of effector lymphocytes in adoptive immunotherapy, although effective in selected solid tumors, suffers on the other hand, from a lack of specificity (such as in the case of lymphokine-activated killer cells (LAK cells) (4) which are mainly NK cells) or from the difficulty in recruiting tumor-infiltrating lymphocytes (TILs) and expanding such specific T cells for most malignancies (5). Yet, the observations that TILs can be obtained in melanoma and renal cell carcinoma tumors, that they can be effective in selected patients and that foreign genes can function in these cells (6) demonstrate the therapeutic potential embodied in these cells.
A strategy which has been recently developed (European Published Patent Application No. 0340793, Ref. 7-11) allows one to combine the advantage of the antibody's specificity with the homing, tissue penetration, cytokine production and target-cell destruction of T lymphocytes and to extend, by ex vivo genetic manipulations, the spectrum of anti-tumor specificity of T cells. In this approach the laboratory of the present inventors succeeded to functionally express in T cells chimeric T cell receptor (cTCR) genes composed of the variable region domain (Fv) of an antibody molecule and the constant region domain of the antigen-binding TCR chains, i.e., the α/β or γ/δ chains. In this gene-pairs approach, genomic expression vectors have been constructed containing the rearranged gene segments coding for the V region domains of the heavy (VH) and light (VL) chains of an anti-2,4,6-trinitrophenyl (TNP) antibody (Sp6) spliced to either one of the C-region gene segments of the α or β TCR chains. Following transfection into a cytotoxic T-cell hybridoma, expression of a functional TCR was detected. The chimeric TCR exhibited the idiotope of the Sp6 anti-TNP antibody and endowed the T cells with a major histocompatibility complex (MHC) nonrestricted response to the hapten TNP. The transfectants specifically killed TNP-bearing target cells, and produced interleukin-2 (IL-2) in response thereto across strain and species barriers. Moreover, such transfectants responded to immobilized TNP-protein conjugates, bypassing the need for cellular processing and presentation. The chimeric TCRs could provide T cells with an antibody-like specificity and, upon encountering antigen, were able to effectively transmit signals for T cell activation, secretion of lymphokines and specific target cell lysis in a MHC nonrestricted manner. Moreover, the cTCR bearing cells undergo stimulation by immobilized antigen, proving that receptor-mediated T-cell activation is not only nonrestricted but also independent of MHC expression on target cells (8, 9). New expression cassettes were also developed based on reverse transcription of mRNA and PCR amplification of rearranged VH and VL DNA, using primers based on 3′ and 5′ consensus sequences (12) of these genes which allow rapid construction of cTCR genes from any mAb-producing hybridoma. To determine the therapeutic potential of the chimeric TCR approach, we successfully constructed and functionally expressed cTCR genes composed of combining sites of anti-idiotypic antibody specific to the surface IgM of the 38C13 murine B lymphoma cell line.
Broad application of the cTCR approach is dependent on efficient expression of the cTCR genes in primary T cells. So far, utilizing protoplast fusion, lipofection or electroporation, we succeeded in expressing the cTCR in T cell hybridomas (8, 9) or human T cell tumors, such as Jurkat, but like others, achieved only limited and transient expression in non-transformed murine T cell lines. Although retroviral vectors have been demonstrated to be effective for transgene expression in human T cells (13, 14), due to the fact that two genes have to be introduced in order to express functional cTCR; (CαVH+CβVL or CαVL+CβVH), and the very low efficiency of transduction of a single cell with two separate retroviral vectors, new vectors have to be tried which will allow the transduction of two genes in tandem (15).
Another strategy which has recently been developed employs joining of the extracellular ligand binding domain of receptors such as CD4, CD8, the IL-2 receptor, or CD16, to the cytoplasmic tail of either one of the γ/ζ family members (26-28, 38). It has been shown that crosslinking of such extracellular domains through a ligand or antibody results in T cell activation. Chimeric CD4 or CD16-γ/ζ molecules expressed in cytotoxic lymphocytes could direct specific cytolysis against appropriate target cells (26, 38). In PCT WO92/15322 it is suggested that the formation of chimeras consisting of the intracellular portion of T cell/Fc receptor ζ, ε or γ chains joined to the extracellular portion of a suitably engineered antibody molecule will allow the target recognition potential of an immune system cell to be specifically redirected to the antigen recognized by the extracellular antibody portion. However, while specific examples are present showing that such activation is possible when the extracellular portion of receptors such as the CD4 receptor are joined to such ζ, η or γ chains, no proof was presented that when a portion of an antibody is joined to such chains one can obtain expression in lymphocytes or activation of lymphocytes.